Blood glucose homeostasis depends on glucose-stimulated insulin secretion (GSIS) from b cells in pancreatic islets. Diminished insulin secretion, along with peripheral insulin resistance, is a hallmark of Type II diabetes. Recent work indicates that glucose signaling depends on subcellular localization of molecules. For instance, biochemical and immunolabeling approaches have shown that glucokinase (GK) is associated with insulin granules. However, these data provide only static snapshots of b cell function and do not elucidate the interplay between signaling pathways involved in GSIS. We have now shown that binding of GK to granules is regulated by b cell insulin receptor and that this regulation correlates with GK activity changes. Other work has shown that while glucose stimulates increases in both cytoplasmic and mitochondrial metabolism, certain nutrients can cause secretion through mitochondrial metabolism alone, yet the mechanisms underlying these different processes is not understood. Intercellular synergy also plays a role in GSIS, since b cells within intact islets secrete approximately 10-fold more insulin in response to glucose than do isolated b cells; this functional enhancement can only be examined in intact islets. Using our unique quantitative optical imaging methods, the dynamics of these subcellular events can be followed accurately in living cells within intact islets. We hypothesize that dynamic subcellular localization of glucose signaling, such as binding of GK to secretory vesicles and compartmentalization in organelles, is required for normal regulation of GSIS. We also hypothesize that gap junctional coupling between b cells in the islet accounts for the increased insulin response from intact islets over isolated b cells. The validity and limits of these hypotheses will be determined via three specific aims: 1) To determine the ability of gap junctional coupling to enhance insulin secretion. 2) To determine the mechanism of GK binding to insulin containing vesicles and the role of this binding in the regulation of GK activity. 3) To determine the relative roles of cytoplasmic and mitochondrial metabolism in GSIS, and determine the ability of each compartment to compensate for defects in the normal signaling pathway. Because numerous studies have shown marked differences between isolated b cells and whole islets, the proposed experiments will be performed largely on intact islets, but guided by parallel investigations using tissue culture models. Experiments on intact islets are made possible through a unique combination of quantitative fluorescence imaging methods and will utilize several available transgenic and tissue-specific knock-out mouse models with demonstrated diabetic phenotypes. Further, we propose a knock-in mouse model containing a GK-GFP fusion that will greatly facilitate the proposed experiments. These novel investigations will advance our understanding of the in vivo interplay between biochemical mechanisms that are directly involved in diabetic phenotypes, and bring us closer to our long-term goal of understanding the spatio-temporal dynamics of glucose-stimulated insulin secretion.